AbstractIntroduction: Diabetes is a disease characterised by chronic hyperglycaemia due to reduced insulin secretion or action. Up to 75% of diabetic patients die due to cardiovascular complications such as heart failure (HF). The term “diabetic cardiomyopathy” describes distinct structural and functional changes in the heart driven by metabolic and microvascular abnormalities. Microvascular dysfunction appears at an early stage as a result of impaired endothelial function, at least in part due to hyperglycaemia-induced production of reactive oxygen species (ROS) in endothelial cells (ECs), leading to oxidative stress and increased vascular tone. NADPH oxidases are enzymes whose primary function is to produce ROS, with NOX2 and NOX4 both highly expressed in ECs and upregulated in diabetic and failing hearts. Our hypothesis was that endothelial-specific NOX2 and NOX4 play a key role in regulating the development of microvascular dysfunction and subsequent progression of diabetic cardiomyopathy.
Methods: Endothelial-specific Nox2- (EC-Nox2Tg) and Nox4-overexpressing (ECNox4Tg) mice were injected with streptozotocin (STZ) to destroy pancreatic β-cells and induce type 1 diabetes (T1D). Some mice were infused with angiotensin (ATII) at a rate of 1.1 mg/kg/day to exacerbate the diabetic cardiac remodelling phenotype. Echocardiography was performed at three and six months of age to identify changes in cardiac structure and function. Assessment of left ventricular (LV) gene and protein expression (qRT-PCR and Western blot), fibrosis (picrosirius red), cardiomyocyte hypertrophy (Haematoxylin and eosin) and total ROS/superoxide (O2 - ) production (DCF/DHE staining) was performed to identify the effects of endothelial-specific Nox2 and Nox4 on diabetic cardiomyopathy.
To assess the effects of hyperglycaemia on EC function, human aortic ECs (HAoECs) with or without NOX4 modification were assessed for changes in gene and protein expression, ROS/O2 - production, intracellular Ca2+ release (Flexstation) and cytokine release (cytokine array) in response to normal glucose (NG; 5.5mM) or high glucose (HG; 25mM). Experiments were repeated in coronary microvascular ECs (CMECs) to study differences in macro- vs. microvascular function. 3T3 fibroblasts and ventricular human cardiac fibroblasts (VHCFs) were treated with conditioned media from ECs to investigate paracrine effects on fibrosis.
Results: While diabetic EC-Nox2 WT mice tended to exhibit decreased diastolic function relative to non-diabetic counterparts, there was no change in cardiac function in control or diabetic EC-Nox2Tg mice. Similarly, there were no differences in LV Nox2 mRNA or protein expression or cardiomyocyte hypertrophy between any of the experimental groups. Moreover, LV fibrotic, hypertrophic, antioxidant or inflammatory gene expression remained largely unaltered. While further experiments are required, these data suggest that endothelial-specific Nox2 overexpression does not exert significant effects on cardiac remodelling and dysfunction in experimental T1D.
Similar to diabetic EC-Nox2WT mice, diabetic EC-Nox4WT mice showed reduced diastolic function relative to non-diabetic counterparts, whilst control EC-Nox4Tg mice showed reduced diastolic function, increased fibrosis, increased O2 - relative to total ROS, and increased expression of fibrotic, antioxidant and inflammatory genes. While the phenotype of EC-Nox4Tg mice was not worsened by STZ diabetes, additional ATII infusion resulted in reduced diastolic and systolic function, increased fibrosis and cardiomyocyte hypertrophy, increased expression of fibrotic, hypertrophic and inflammatory genes and reduced expression of antioxidant genes. Taken together, these data suggest that endothelial-specific Nox4 overexpression is a key driver of diabetic cardiomyopathy.
Complementary in vitro investigation showed that HG increased NOX4 expression in both HAoECs and CMECs. NOX4 overexpression in HAoECs led to increased antioxidant gene expression, altered ROS production, increased Ca2+ signalling and protein phosphorylation, and increased cytokine release in response to HG. Both 3T3 fibroblasts and VHCFs showed exacerbated fibroblast differentiation following treatment with conditioned media from HG-treated NOX4-overexpressing HAoECs. Nox4 overexpression in CMECs exacerbated protein phosphorylation and increased expression of inflammatory, antioxidant, angiogenic and fibrotic genes, whilst conditioned media from HG-treated Nox4 overexpressing CMECs exacerbated differentiation of VHCFs. These data suggest that endothelial Nox4 is central to HGinduced pathological signalling via alteration of ROS production and consequent increases in inflammation and fibrosis which seems to be more severe in the microvasculature.
Conclusions: We concluded that endothelial-specific Nox2 overexpression likely has limited effects on T1D diabetic cardiomyopathy, although it would be interesting to compare with type 2 diabetes (T2D) in which the cardiac phenotype is different. However, endothelial-specific Nox4 overexpression appears to play a key role in the development and progression of diabetic cardiomyopathy in experimental T1D via specific effects on ROS and pro-fibrotic signalling. As such, it is likely that functional ~ vi ~ changes typically observed in the diabetic heart are at least partly due to pathological Nox4-mediated changes in endothelial ROS production and signalling, leading to increased cytokine release, fibrosis, and adverse remodelling. Therefore, targeting endothelial-specific Nox4 in the cardiac microvasculature may represent a novel therapeutic approach to slow progression of diabetic cardiomyopathy.
|Date of Award
|Northern Ireland Department for the Economy & British Heart Foundation
|David Grieve (Supervisor) & Tim Curtis (Supervisor)